In the last decade there has been an explosive growth in the field of ubiquitin research, with approximately 530 human genes predicted to encode enzymes involved in the conjugation and deconjugation of ubiquitin. Of these 95 encode for deubiquitylases (DUBs). In order understand the biology of these enzymes better, there is a need for better assays to measure the most physiologically relevant activity of the enzymes. All the currently available high throughput methods for measuring deubiquitylase activity rely on C-terminal amidohydrolase activity (involved in processing the precursors of ubiquitin) rather than the isopeptidase activity involved in ubiquitin deconjugation (important in regulating various cellular processes) of DUBs. The most widely used substrate, Ub-AMC has a small fluorophore attached to the C-terminus of ubiquitin via an amide bond. Hydrolysis of this C-terminal amide bond by a deubiquitylase leads to an increase in fluorescence. This assay format does not adequately mimic the more important physiological event - deconjugation of ubiquitin via isopeptidase activity. Furthermore, many DUBs do not possess C-terminal amidohydrolytic activity and hence are unable to cleave conjugates like Ub-AMC. Although it is possible to measure isopeptidase activity with physiological substrates such as commercially available ubiquitin chains by SDS-PAGE, western blotting or LC/MS, such options are viable only if a small number of samples are being tested. For screening small molecules or natural products for inhibitors of isopeptidases these methods are unacceptable. In Phase I we developed a novel fluorescent assay for measuring the actual isopeptidase activity of the DUBs with substrates that are more relevant to physiological conditions. This assay is amenable to high throughput screening and does not suffer from the limitations shared by current DUB assays. Briefly, we have created a series of diUb molecules in which one Ub chain is derivatized with a fluorescence quenching dye and the second Ub moiety carrys a fluorophore. The two Ubs are joined by an isopeptide bond linking the C-terminus of one Ub to either Lys48 (K48) or Lys63 (K63) of the second Ub. Following hydrolysis of this isopeptide bond by a DUB, FRET-quenching is released and the resulting increase in fluorescence is directly proportional to DUB activity. We have validated these substrates using DUBs which only cleave the K48- or K63-linkage. In the current grant application, we propose to extend this technology to the remaining 5 Lys's in ubiquitin and create a panel of substrates encompassing all of ubiquitin isopeptide linkages. These substrates will greatly enhance our understanding of DUB activity and selectivity and enable high throughput screening campaigns using physiologically relevant substrates.